Rotavirus (RV) belongs to the rotavirus genus belonging to the Reoviridae family, which is the main pathogen responsible for infant diarrhea and was found in duodenum from patients with gastroenteritis by Bishop in 1973 (Bishop, Davidson, Holmes, et al. Lancet, 2 (7841), 1281-1283, 1973). Studies showed that more than 95% of children were infected with rotavirus at least once before 5 years old. According to the WHO statistics, up to 600,000 people died of rotavirus infection annually, and cases of diarrhea reached up to 200 million; and in USA only, economic loss caused by rotavirus infection reached up to 1 billion dollars annually (Hsu, Staat, Roberts, et al. Pediatrics, 115 (1), 78-82, 2005; Tate, Burton, Boschi-Pinto, et al. The Lancet Infectious Diseases, 12 (2), 136-141, 2011), resulting in serious financial burden and social burden.
Rotavirus is a non-enveloped RNA virus, and its genome consists of 11 double-stranded RNA molecules which encode 6 structural proteins (VP1-VP4, VP6 and VP7) and 6 non-structural proteins (NSP1-NSP6) (Estes and Cohen. Microbiol Rev, 53 (4), 410-449, 1989). Rotavirus is icosahedral, and its capsid consists of three concentric layers, i.e. the core layer consisting of VP1, VP2 and VP3, the inner capsid consisting of VP6, and the outer capsid consisting of VP4 and VP7.
VP6 protein is a capsid protein comprised in the highest content in rotavirus. Depending on the antigenicity of VP6, rotavirus may be divided into 7 groups, i.e. rotavirus A-G, among which rotavirus A is the main pathogen responsible for diarrhea among infants and young children. VP4 and VP7 are the main neutralizing antigens, and, rotavirus A can be divided into P serotype and G serotype depending on their antigenicity. G serotype and P serotype are independent of each other but are also interacted with each other; the common combinations include G1P[8], G2P[4], G3P[8] and G4P[8]; in recent years, the prevalence of G9P[8] and G9P[6] is significantly increased (Li, Liu, Yu, et al. Vaccine, 27 F40-F45, 2009).
There are not specific drugs for rotavirus yet, and safe and effective vaccines are the important means for control of rotavirus infection. After years of research, the vaccine development has underwent three phases, i.e. monovalent attenuated vaccines, polyvalent gene recombinant vaccines, and genetically engineering vaccines. At present, there are five rotavirus vaccines appeared in the market one by one, including tetravalent human-ape gene recombinant vaccine from Wyeth, monovalent attenuated vaccine from Lanzhou Institute, pentavalent human-bovine gene recombinant vaccine from Merck, monovalent attenuated vaccine from GSK, and monovalent attenuated vaccine from Bharat Vaccines, India. However, these vaccines are attenuated live vaccines which have large potential safety hazard, and among them, the vaccines from Wyeth were recalled due to intestinal intussusception half a year after being in the market (Murphy, Gargiullo, Massoudi, et al. 344 (8), 564-572, 2001). Although the vaccines from MERCK and GSK were demonstrated to be safe and effective by a large number of clinical tests (Bernstein, Sack, Rothstein, et al. Lancet(British edition), 354 (9175), 287-290, 1999; Vesikari, Matson, Dennehy, et al. New England Journal of Medicine, 354 (1), 23-33, 2006; Linhares, Velázquez, Pérez-Schael, et al. Lancet, 371 (9619), 1181-1189, 2008; Vesikari, Itzler, Karvonen, et al. Vaccine, 28 (2), 345-351, 2009; Snelling, Andrews, Kirkwood, et al. Clinical Infectious Diseases, 52 (2), 191-200, 2011), in countries and regions with a high rotavirus mortality such as Asia and Africa, the protection efficiency was much lower than that in developed countries such as Europe and America (Armah, Sow, Breiman, et al. Lancet, 376 606-614, 2010; Zaman, Anh, Victor, et al. Lancet, 376 568-570, 2010; Madhi, Cunliffe, Steele, et al. N Engl J Med, 362 (4), 289-298, 2011). More and more evidence showed that upon vaccination with the two vaccines, excretion of virus occurred, which might cause horizontal transmission of virus (Anderson. Lancet Infect Dis, 8 (10), 642-9, 2008; Rivera, Pena, Stainier, et al. Vaccine, 29 (51), 9508-9513, 2011; Yen, Jakob, Esona, et al. Vaccine, 29 (24), 4151-5, 2011). It was also shown in some studies that serious gastroenteritis might be developed in immune-deficient children after vaccination with the vaccines (Steele, Cunliffe, Tumbo, et al. J Infect Dis, 200 Suppl 1 S57-62, 2009; Patel, Hertel, Estes, et al. N Engl J Med, 362 (4), 314-9, 2010). The vaccines from Lanzhou Institute have been commercially available for more than 10 years, and no serious problem is found yet; however, they can only prevent serious diarrhea, but cannot prevent rotavirus infection (Fu, Wang, Liang, et al. Vaccine, 25 (52), 8756-61, 2007). Therefore, although delightful results are obtained in the studies on attenuated vaccines against rotavirus, there are still some problems, and the safety and effectiveness need to be further improved. It is imperative to develop more safe and effective vaccines.
Non-replicating vaccines are the main direction for studies on rotavirus vaccines now, and genetically engineering vaccines attract much attention because of characteristics such as low cost, safety and effectiveness.
VP4 and VP7 protein, as neutralizing antigens of rotavirus, can stimulate generation of neutralizing antibodies in an organism, thereby inhibiting infection of rotavirus. Therefore, both VP4 and VP7 are the main candidate antigens for rotavirus subunit vaccines. VP7 protein, which is a glycosylated protein, comprises 4 intrachain disulfide bonds, and forms a Ca2+-dependent trimer, but the level of neutralizing antibodies, generated by stimulation with a recombinantly expressed VP7 protein, is low. VP4 protein is not glycosylated, and a recombinantly expressed VP4 protein can stimulate generation of high immune response in an organism, and reduce the degree of diarrhea in suckling mice (Mackow, Vo, Broome, et al. J Virol, 64 (4), 1698-703, 1990). In another aspect, there are only two common P serotypes, but there are five G serotypes. Therefore, as compared to VP7 protein, VP4 protein is a candidate antigen more suitable for rotavirus genetic engineering vaccines.
VP4 protein consists of 776 amino acids, can be cleaved by trypsin to form two moieties, i.e., VP8* (aa1-231) and VP5* (aa248-776). VP4 protein consists of a head domain, a body and stalk domain and a foot domain, and as a spike protein of rotavirus, VP4 plays an important role in rotavirus infection. The head domain of a spike consists of two molecules of VP8 protein, and VP8 protein can bind to sialic acid receptor on the surface of a cell, and thereby mediate the adsorption of rotavirus. The body and stalk domain consists of three molecules of VP5 antigen domain, wherein two molecules of VP5 antigen domain form a dimer, and another VP5 antigen domain is present in a form of monomer. During the infection of rotavirus, the structure of VP4 is rearranged, to expose the membrane fusion site in VP5 and mediate the entry of rotavirus into a cell, during which, VP5 antigen domains change from a dimer to a trimer. The foot domain consists of three molecules of C-terminal domain of VP5 protein, and inserts into outer capsid and inner capsid by virtue of the domain. The first 25 Amino acids at N-terminal of VP8 protein form an α-helix, three α-helixes form a helical bundle that inserts into VP5 foot domain, and is linked to a VP8 head domain via a flexible jointing region, wherein the structure of the jointing region is not completely determined yet (Settembre, Chen, Dormitzer, et al. EMBO J, 30 (2), 408-16, 2011).
There are studies showing that neutralizing epitopes of VP4 protein are mainly present in VP8 head domain and VP5 antigen domain, neutralizing antibodies against VP8 can inhibit the absorption of rotavirus, and neutralizing antibodies against VP5 have cross-neutralizing activity, and can inhibit the entry of rotavirus into a cell (Dormitzer, Nason, Venkataram Prasad, et al. Nature, 430 (7003), 1053-1058, 2004; Abdelhakim, Salgado, Fu, et al. PLoS Pathog, 10 (9), e1004355, 2014). In addition, the results of scanning synthetic peptides show that there are also neutralizing epitopes in a-helical region at N-terminal and the jointing region of VP8 protein (Kovacs-Nolan, Yoo and Mine. Biochem J, 376 (Pt 1), 269-75, 2003).
There are a lot of research results showing that VP4 protein can stimulate protective immune response in an organism (Dunn, Fiore, Werner, et al. Arch Virol, 140 (11), 1969-78, 1995; Gil, De Souza, Asensi, et al. Viral Immunology, 13 (2), 187-200, 2000). However, expression of VP4 protein by eukaryotic system has the disadvantages of long cycle, high cost and low expression level; in addition, a full-length VP4 protein is mainly present in a form of inclusion body in prokaryotic system, is difficult to purify and cannot retain its natural conformation. Wen Xiaobo et al. solved the problem concerning the expression of VP8 protein in a form of inclusion body in prokaryotic system by means of expressing a truncated form thereof, and obtained the truncated VP8 protein (ΔVP8*, aa65-223) that could be effectively expressed in E. coli in a soluble form (Wen, Cao, Jones, et al. Vaccine, 30 (43), 6121-6, 2012); however, the immunogenicity of the truncated VP8 protein is significantly lower than the full-length VP8 protein. In order to solve the problem concerning the low immunogenicity of the truncated VP8 protein, after deep studies, the laboratory of the inventor obtained new truncated VP8 proteins with higher immunogenicity, which can stimulate generation of high-titer neutralizing antibodies in mice upon the immunization therewith in the presence of Freund's adjuvant, and can reduce the degree of diarrhea in suckling mice; and the truncated VP8 protein can be subjected to fusion expression with an intramolecular adjuvant, so as to produce a fusion protein with higher immunogenicity and immune-protection (CN201510165746.2). Although the new truncated VP8 proteins and the fusion protein thereof have achieved significantly advantageous technical effects, there are still shortcomings. For example, antibodies against VP8 protein are mainly serotype-specific antibodies, and therefore, the new truncated VP8 proteins as vaccines have a significantly lower neutralizing activity against virus strains of a different serotype than the neutralizing activity against virus strains of the same serotypes (Wen, Cao, Jones, et al. Vaccine, 30 (43), 6121-6, 2012). In addition, it is also found in the subsequent studies that the immune-protection of the new truncated VP8 proteins is not satisfactory enough in the presence of aluminum adjuvant, and needs to be further improved.
Therefore, there is still demand in the art to develop new vaccines against rotavirus, which can be produced more conveniently in a high expression level (e.g., by means of soluble expression and purification), can have stronger immunogenicity than the existing vaccines (e.g., the truncated VP8 protein mentioned above), can induce generation of high-titer neutralizing antibodies against rotavirus (particularly in the presence of aluminum adjuvant) in an organism, and therefore can be used in the large-scale industrial production of highly effective vaccines against rotavirus.